The ciliated cells coating the apical surface of the epithelia are essential for various physiological processes such as cleaning of the respiratory passages, embryo implantation, or circulation of the cerebrospinal fluid. Defective ciliogenesis is the direct cause of or is associated with a great variety of diseases.
The process of ciliogenesis comprises a sequence of events that begins with acquisition of the identity of the ciliated cell (phase 1). This first step consists of lateral inhibition between two adjacent cells by the Notch signalling system via interaction between Notch and its ligand such as delta-like 1 (DLL1). The cell expressing the DLL1 ligand becomes a progenitor cell of ciliated cells, and simultaneously, activation of Notch in the neighbouring cells prevents transformation of these cells into progenitor cells of ciliated cells. The inventors have shown that the progenitor cell of ciliated cells expresses the microRNAs of the family miR-449 and the transcription factor FOXJ1. During a second phase, the miR-449s inhibit cell division and induce differentiation. Multiplication of the centrioles begins in the progenitor cell of ciliated cells, and this multiplication is followed by anchoring of the basal bodies to the apical pole of the cells; this step is followed by assembly of the axoneme and cilia synthesis proper.
The epithelia perform a barrier function between the internal medium and the external environment. The respiratory passages are coated with a highly differentiated pseudostratified epithelium consisting of mucus-secreting and ciliated basal cells (each ciliated cell having hundreds of cilia). The coordinated movement of these numerous cilia present on the surface of the epithelium permits the removal of waste carried by the mucus during a process called mucociliary clearance. In this connection, the cilia play an important role in the processes of defense against first-line respiratory tract infections (Puchelle et al. Proc Am Thorac Soc (2006) 3, 726-733).
The permanent exposure of the airway epithelium to environmental stresses caused by pathogenic microorganisms, allergens, toxic molecules, etc. leads to tissue lesions. Following these lesions, a physiological process of regeneration of the airway epithelium goes into action. This process, when successful, repairs the lesion and restores the integrity of the respiratory tissue, the lesion being replaced with tissue that is differentiated and is functional again.
This regeneration comprises several steps:
1) the epithelial cells proliferate and/or migrate in order to fill the wound bed;
2) these first steps are followed by activation of a step of cellular polarization characterized by the formation of tight junctions and by specific differential addressing of membrane proteins (channels, ion transporters etc.) between the apical pole and the basolateral pole (Puchelle et al. 2006; Hajj, R. et al. J Pathol (2007) 211, 340-350);
3) a stage of terminal differentiation leading to the formation of cilia on the surface of the ciliated cells (ciliogenesis) and to the presence of secretory cells responsible for the synthesis and secretion of mucus.
A pseudostratified mucociliary epithelium thus replaces the lesion, reconstituting a functional ciliated tissue having properties identical to those of the original tissue.
Taken together, these biological phenomena are associated with mechanisms of signal transduction and with particular gene expression profiles. Among certain known actors implicated in the differentiation and ciliogenesis of the airway epithelium, the Foxj1 transcription factor is one of the best documented (Yu, X. et al. (2008) Nat Genet. 40, 1445-1453). Foxj1 acts in a late phase of ciliogenesis, playing a role in anchoring of the basal bodies (small organelles of structural organization close to the centrioles that are indispensable for formation of the base of the cilia) to the apical membrane during formation of the axoneme (Gomperts, B. N. et al. (2004) J Cell Sci 117, 1329-1337 (2004).
In certain chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD), mucoviscidosis, asthma or primary ciliary dyskinesia (PCD), inflammations and chronic infections lead to destruction of the respiratory tissue (Marshall, W. F. (2008) J Cell Biol 180, 17-21). For reasons that are still poorly understood, these diseases are associated with defects of epithelial regeneration and differentiation. These defects result in abnormal restructuring of the tissue, fibrosis and irreversible functional loss (Marshall, W. F., 2008). There is still no therapeutic treatment for these various diseases, and only symptomatic treatments are available for combating, with a varying degree of effectiveness, the progressive destruction of the respiratory tissue. In this connection, elucidation of the mechanisms leading to the formation of functional cilia (ciliogenesis) represents a major challenge with obvious therapeutic benefits.
Cellular differentiation involves fine temporal and spatial regulation of the transcription and translation governing the expression of specific genes. These events are controlled by various molecular and mechanical signals. Understanding the physiological mechanisms underlying differentiation and ciliogenesis is therefore an indispensable prelude to the development of therapeutic approaches that are more specific and more effective.
The microRNAs (miRNAs), small noncoding RNAs of about 22 bases discovered in 1993, which have regulatory properties, play a key role in the regulation of cellular phenomena such as survival, apoptosis, proliferation, homeostasis or differentiation (Lu, Y. et al. (2007) Dev Biol 310, 442-453).
Their mechanisms of action involve the formation of a complex between several bases of the miRNA and the noncoding 3′ portion of the target mRNA. This interaction is said to induce destabilization of the target mRNA and/or inhibition of protein synthesis. Recognition between a miRNA and its target is mainly controlled by a sequence of about 7 bases, situated in the 5′ portion of the miRNA (hereinafter, recognition sequence or seed). Accordingly, each miRNA would have the capacity to regulate the stability of a wide range of separate mRNAs.
To date, more than 750 miRNAs have been characterized in humans, where they are said to regulate more than 30% of the transcripts. Regulation by the miRNAs therefore appears to be a major regulation of gene expression, the impact of which has been underestimated until now (Berezikov, E. et al. (2005) Cell 120, 21-24; Xie, X. et al. (2005) Nature 434, 338-345).
The miRNAs are transcribed in the nucleus in the form of long precursors. They undergo a first maturation in the nucleus to give a precursor of miRNA (pre-miRNA) possessing a smaller hairpin structure. This precursor is exported from the nucleus to the cytoplasm where it will undergo a final maturation. Degradation of its loop by the enzyme Dicer generates two single-stranded miRNAs (a 5p strand and a 3p strand); the so-called mature strand is managed by a multi-protein complex (the RISC complex: RNA induced silencing complex) which interacts with the noncoding 3′ portion of the target mRNAs, whereas the so-called “star” complementary strand will undergo a degradation; the complementary strand of a miRNA miR-xy, miR-xy-z or let-7x is designated, respectively, miR-xy*, miR-xy-z* or let-7x*.
Recent studies have demonstrated the importance of the microRNAs in the mouse in the mechanisms of differentiation and morphogenesis; in particular in embryonic development and the proliferation of the precursors of the epithelial cells of the epidermis (Lena, A. M. et al. (2008) Cell Death Differ 15, 1187-1195; Yi, R. et al. (2008) Nature 452, 225-229) or in lung development (Lu, Y. et al. (2007) Dev Biol 310, 442-453 (2007); Harris, K. S. et al. (2006) Proc Natl Acad Sci USA 103, 2208-2213; Lu, Y. et al. (2008) Proc Am Thorac Soc 5, 300-304). More precisely, Lena et al. demonstrated the involvement of the miRNAs of the locus miR-17-92 in pulmonary morphogenesis in the mouse. Although there is proof of the involvement of miRNAs during pulmonary morphogenesis in the mouse, their precise role(s) and their mechanisms of action have yet to be investigated.
Finally, several studies suggest a particular role of certain miRNAs in diseases such as cancers, cardiac hypertrophy, diabetes or certain viral infections (Triboulet, R. et al. (2007) Science 315, 1579-1582; Calin, G. A. & Croce, C. M. (2006) Nat Rev Cancer 6, 857-866; Grassmann, R. & Jeang, K. T. (2008) Biochim Biophys Acta 1779, 706-711; Latronico, M. V., (2008) Physiol Genomics 34, 239-242; Poy, M. N. et al. (2004) Nature 432, 226-230).
To date, no study has demonstrated the role or involvement of miRNAs in the regeneration and differentiation of ciliated epithelia, such as the airway epithelium, and the control of ciliogenesis in vertebrates.